Studies of the induction of anti-DNA in normal mice

Injection of PBA has previously been demonstrated to induce anti-DNA. In the present study, we found that the combination of neonatal thymectomy and chronic administration of PBA (LPS + poly rI . rC) led to significantly higher anti-DNA levels than either PBA or thymectomy separately. These results suggested that a thymic regulatory process normally serves to suppress anti-DNA after chronic PBA exposure. Indeed, antigen-nonspecific suppressor function was found to be deficient in such thymectomy + PBA-treated mice. In addition, the cells of such mice in vitro interfered with the development of normal suppressor function by control cells.     

Intercellular junctions in the uterine epithelium of Salamandra salamandra (L.) (Amphibia,Urodela)

Summary Intercellular junctions in the uterine epithelium of the ovoviviparous urodele Salamandra salamandra were studied in pregnant and non-pregnant females by freeze-fracture technique. Junctional complexes consist of zonulae occludentes (tight junctions) and numerous maculae adhaerentes (desmosomes); z. adhaerentes and nexuses (gap junctions) could not be identified. Tight junctions are of the flexible type exhibiting loosely interconnected fibrils. The fibrillary network appears stretched more often in pregnant females possibly due to the mechanical stress of pregnancy. The structure and occurrence of the junctions identified, especially that of the tight junctions, is discussed with regard to the functions of the uterus during pregnancy.Zusammenfassung Mit Hilfe der Gefrierbruchtechnik wurden im Uterus-epithel trächtiger und nichtträchtiger Feuersalamanderweibchen (Salamandra salamandra) Zonulae occludentes und Maculae adhaerentes, jedoch keine Z. adhaerentes sowie Nexus identifiziert. Die Z. occludentes sind flexibel. Ihr fibrilläres Netzwerk ist bei trächtigen Weibchen öfter gestreckt; das ist möglicherweise auf die stärkere Ausdehnung des Uterusgewebes während der Trächtigkeit zurückzuführen. Das Vorkommen der verschiedenen Kontakt-strukturen, namentlich das der Z. occludentes, wird im Hinblick auf die Funktionen des Uterus während der Trächtigkeit diskutiert.We are indebted to Mrs. K. Ott for excellent technical assistance and to Miss Dr. U. Beigel for linguistic help     

Clofilium1--a new antifibrillatory agent that selectively increases cellular refractoriness

Clofilium is the most promising member in a new series of antifibrillatory agents to selectively prolong cardiac action potential duration (APD) and effective refractory period (ERP). In normal superfused canine Purkinje fibers, clofilium prolonged APD and ERP by a maximum of 35% (ED₅₀=1.3 × 10^?8 M). The effect of clofilium reached equilibrium in 61±3 min but APD did not return toward control during several hr of superfusion with drug-free medium. No change in rate of rise, amplitude, resting potential or rate of diastolic depolarization was noted in the presence of clofilium (3 × 10^?7 M). Clofilium increased the canine ventricular fibrillation threshold (VFT) measured using gated trains of electrical stimuli. This effect occurred in a dose-related fashion following a 30 min infusion of a total of 0.5 or 1.0 μmole/kg of clofilium. The increase was evident within 30 min after ending the infusion and persisted for at least 4 hr. Following the infusion of clofilium (1.0 μmole/kg) 22% of the episodes of ventricular fibrillation (VF) spontaneously reverted to normal sinus rhythm without the use of direct current countershock; this phenomenon did not occur in dextrose-infused dogs.     

F plasmid replication and the division cycle of Escherichia coli B/r

A terminal stage in the duplication of many bacterial plasmids involves the transient formation of catenated molecules containing two interlocked monomeric plasmid units. This property of plasmid replication was exploited to examine the relationship between F replication and the division cycle of Escherichia coli B/r cells growing in undisturbed, exponential-phase cultures. Various cultures of F′lac- or FKm^r-containing cells were briefly exposed to [³H]thymidine, and then the transfer of radioactivity into, and out of, a catenated dimer consisting of two closed circular monomers was measured during a chase period. The fraction of plasmid molecules present in this dimer form was determined by separating cellular DNA in alkaline sucrose gradients. In addition, plasmid replication was studied in synchronously growing cultures by measuring both [³H]thymidine incorporation into covalently closed circular DNA and β-galactosidase inducibility. The results suggest that replication of F plasmids can take place throughout the cell division cycle, with the probability of replication increasing toward the end of the cycle. The presence of DNA homologous to the chromosome on the F′lac did not alter the replication pattern of the plasmid during the division cycle.     

Mutations causing charge alterations in regulatory subunits of the cAMP-dependent protein kinase of cultured S49 lymphoma cells.

Two-dimensional polyacrylamide gel electrophoresis is used to visualize the regulatory subunit of cAMP-dependent protein kinase from cultured S49 mouse lymphoma cells and to demonstrate its in vivo phosphorylation. Regulatory subunits from mutant cells with altered kinases exhibit at least two patterns of charge shifts consistent with substitutions of single amino acids. The direct demonstration of structural alteration of this protein provides strong evidence for structural gene mutation in this cultured cell system. While mutant and wild-type gene products co-exist in the mutant cells, there is apparently preferential expression and phosphorylation of mutant subunit in these heterozygotes.     

Uptake and degradation of high density lipoprotein: comparison of fibroblasts from normal subjects and from homozygous familial hypercholesterolemic subjects.

Fibroblasts cultured from the skin of subjects with homozygous familial hyperlipoproteinemia (HFH) internalize and degrade low density lipoproteins at a much lower rate than do fibroblasts from normal subjects. Evidence has been presented that this reflects the absence from such mutant cells of specialized binding sites with high affinity for low density lipoproteins. The specificity of this membrane defect in familial hypercholesterolemia is further supported by the present studies comparing the metabolism of low density lipoproteins (LDL) and high density lipoproteins (HDL) in normal fibroblasts and in fibroblasts from HFH patients. The surface binding (trypsin-releasable (125)I) of (125)I-labeled LDL by HFH cells was approximately 30% of that by normal cells at a concentration of 5 micro g LDL protein per ml. At the same concentration the internalization (cell-associated (125)I after trypsinization) and degradation (trichloroacetic acid-soluble non-iodide (125)I) of (125)I-labeled LDL were less than 10% of the values obtained with normal cells. In contrast, the binding of (125)I-labeled HDL to HFH cells was actually somewhat greater than that to normal cells. Despite this, the internalization and degradation of (125)I-labeled HDL by HFH cells averaged only 70% of that by normal cells. [(3)H]- or [(14)C]Sucrose uptake, a measure of fluid uptake by pinocytosis, was similar in normal and HFH fibroblasts. These findings are consistent with the proposal that fibroblasts from subjects with HFH lack high-affinity receptors for LDL. These receptors do not play a significant role in HDL binding and uptake. Instead, as previously proposed, HDL appears to bind randomly on the cell surface and its internalization is not facilitated by the specific mechanism that internalizes LDL. The small but significant abnormalities in HDL binding and internalization, however, suggest that there may be additional primary or secondary abnormalities of membrane structure and function in HFH cells. Finally, the observed overall rate of uptake of LDL (that internalized plus that degraded) by HFH fibroblasts was considerably greater than that expected from fluid endocytosis alone. This implies that adsorptive endocytosis, associated with binding to low-affinity sites on the cell surface, may play a significant role in LDL degradation by HFH cells, even though it does not regulate endogenous cholesterol synthesis in these cells.     

Effect of intracellular potassium upon the electrogenic pump of frog retinal pigment epithelium

Summary We have studied the hyperpolarizing, electrogenic pump located on the apical membrane of the retinal pigment epithelium (RPE) in anin vitro preparation of bullfrog RPE-choroid. Changes in RPE [K⁺] _i alter the current produced by this pump. Increasing [K⁺] _o in the solution perfusing thebasal membrane increases RPE [K⁺] _i (measured with a K⁺-specific microelectrode), and also depolarizes theapical membrane. This depolarization is due to a decrease in electrogenic pump current flowing across the apical membrane resistance, since it is abolished when the pump is inhibited by apical ouabain, by cooling the tissue, or by 0mm [K⁺] _o outside the apical membrane. Removal of Cl^– from the solution perfusing the basal membrane abolishes the K⁺-evoked apical depolarization by preventing the entry of K⁺ (as KCl) into the cell. We conclude that the increase in [K⁺] _i causes the decrease in pump current. This result is consistent with the finding that [K⁺] _i is a competitive inhibitor of the Na⁺–K⁺ pump in red blood cells.It is possible that the light-evoked changes in [K⁺] _o in the distal retina could alter RPE [K⁺] _i , and thus could affect the pump from both sides of the apical membrane. Any change in pump current is likely to influence retinal function, since this pump helps to determine the composition of the photoreceptor extracellular space.     

Kinase-negative mutants of S49 mouse lymphoma cells carry a trans-dominant mutation affecting expression of cAMP-dependent protein kinase.

Kinase-negative mutants of S49 mouse lymphoma cells are pleiotropically negative for all known cAMP-mediated responses of S49 cells and yield cell extracts which are deficient in cAMP binding activity and devoid of cAMP-dependent protein kinase activity. In hybrids between kinase-negative and wild-type cells, the mutant phenotype is dominant: the tetraploid hybrids have reduced cAMP-binding activity and undetectable cAMP-dependent kinase activity. The mutant phenotype is attributable to neither a soluble inhibitor of kinase catalytic subunit, nor a defective kinase regulatory subunit acting as an inhibitor, nor a defective catalytic subunit which sequesters regulatory subunits in inactive complexes. We propose that these mutants carry trans-dominant lesions in a regulatory locus responsible for setting intracellular levels of kinase expression.     

Comparison of detergent-solubilized and membrane-bound forms of kidney (Na + K)-ATPase

The detergent solubilization of dog kidney (Na + K)-ATPase has been investigated. The nonionic detergents, Brij 58, C₁₂E₈, and Lubrol WX were tested for their ability to produce active, soluble enzyme. Lubrol WX gave the best results. Enzyme so treated is found in the supernatant fraction after centrifugation at 100,000g for 1 h. It has the same or slightly greater specific activity, the same subunit composition as judged by SDS-gel electrophoresis, and very similar kinetic parameters with respect to sodium, potassium, ATP, pNPP, and ouabain as the membrane-bound enzyme. The Lubrol-treated enzyme is stable for at least 5 days at 4 °C. The phospholipid content of the Lubrol-treated enzyme is decreased, as might be expected, by about 50%. Limited tryptic proteolysis and fluorescence changes seen after modification with FITC indicate that the solubilized (Na + K)-ATPase undergoes the same conformational transitions as the membrane enzyme. Our results indicate that kidney enzyme solubilized as described here is nondenatured and fully active, and therefore a valuable preparation for spectroscopic and other approaches for study of this enzyme.